Image scanning fluorescence emission difference microscopy based on a detector array

Abstract

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.

Resolution Enhancement and Background Suppression in Optical Super‐Resolution Imaging for Biological Applications

“…Top: subtraction of signals obtained by original outer ring detectors from pixel reassigned signals of outer ring detectors. [ 91 ] Bottom: subtraction of the image acquired by doughnut beam from the image by Gaussian beam via detector array imaging. [ 93 ] b) Virtual supercritical angle fluorescence microscopy (vSAF).…”

“…In DA microscopy, like Airyscan [ 89 ] or virtual k ‐space modulation optical microscopy (VIKMOM), [ 90 ] a detector array is able to collect signals at the peripheral regions of the Ariy‐disk and can solve the pile‐up effect caused by the dead time of electronics. [ 91 ] The resolution‐improved confocal microscope based on array detection and photon reassignment has been extended to commercial application in biomedical research and clinical diagnosis.…”

“…Therefore, the subtraction of the two reconstructed images will elicit a sharper PSF and higher spatial frequency (Figure 2a). [ 91 ] Moreover, subtraction of the original signals obtained by the detectors in the outer ring from the reassigned signals could similarly increase the final spatial resolution. In addition, according to previous reports, shrinking the pinhole size will further sharpen the size of the overall 3D PSF.…”

Characterization of neoplastic cells outlining the cystic space of invasive micropapillary carcinoma of the canine mammary gland.

“…A Zeiss LSM with an Airyscan detector was used to overcome the limited image resolution and improve the signal-to-noise ratio compared with those of conventional light microscopes and confocal systems. This system utilizes deconvolution by properly weighting the signals from a combination of 32 detectors to improve the three-dimensional (3D) resolution and has been applied in biomedical research and clinical diagnosis [41]. Observation of immunohistochemical staining does not allow precise colocalization of signals, and multiple colors cannot be visualized simultaneously in 3D [42].…”

Characterization of neoplastic cells outlining the cystic space of invasive micropapillary carcinoma of the canine mammary gland

“…A Zeiss LSM 880 with an Airyscan detector was used to overcome the limited image resolution and improve the signal-to-noise ratio compared with those of conventional light microscopes and confocal systems. This system utilizes deconvolution by properly weighting the signals from a combination of 32 detectors to improve the three-dimensional (3D) resolution and has been applied in biomedical research and clinical diagnosis [25]. Observation of immunohistochemical staining does not allow precise colocalization of signals, and multiple colors cannot be visualized simultaneously in 3D [26].…”